Mutations in GAS8, a Gene Encoding a Nexin‐Dynein Regulatory Complex Subunit,Cause Primary Ciliary Dyskinesia with Axonemal Disorganization

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few—including CCDC39 and CCDC40—carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi‐allelic loss‐of‐function mutations in GAS8, a gene that encodes a subunit of the nexin‐dynein regulatory complex (N‐DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high‐speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients’ respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N‐DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N‐DRC integrity and in the proper alignment of axonemal microtubules in humans.     

Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.     

Induction of chondrogenesis in neural crest cells by mutant fibroblast growth factor receptors.

Activating mutations in human fibroblast growth factor receptors (FGFR) result in a range of skeletal disorders, including craniosynostosis. Because the cranial bones are largely neural crest derived, the possibility arises that increased FGF signalling may predispose to premature/excessive skeletogenic differentiation in neural crest cells. To test this hypothesis, we expressed wild-type and mutant FGFRs in quail embryonic neural crest cells. Chondrogenesis was consistently induced when mutant FGFR1-K656E or FGFR2-C278F were electroporated in ovo into stage 8 quail premigratory neural crest, followed by in vitro culture without FGF2. Neural crest cells electroporated with wild-type FGFR1 or FGFR2 cDNAs exhibited no chondrogenic differentiation in culture. Cartilage differentiation was accompanied by expression of Sox9, Col2a1, and osteopontin. This closely resembled the response of nonelectroporated neural crest cells to FGF2 in vitro: 10 ng/ml induces chondrogenesis, Sox9, Col2a1, and osteopontin expression, whereas 1 ng/ml FGF2 enhances cell survival and Sox9 and Col2a1 expression, but never induces chondrogenesis or osteopontin expression. Transfection of neural crest cells with mutant FGFRs in vitro, after their emergence from the neural tube, in contrast, produced chondrogenesis at a very low frequency. Hence, mutant FGFRs can induce cartilage differentiation when electroporated into premigratory neural crest cells but this effect is drastically reduced if transfection is carried out after the onset of neural crest migration.     

Magnetic resonance elastography with guided pressure waves

Magnetic resonance elastography aims to non-invasively and remotely characterize the mechanical properties of living tissues. To quantitatively and regionally map the shear viscoelastic moduli in vivo, the technique must achieve proper mechanical excitation throughout the targeted tissues. Although it is straightforward, ante manibus, in close organs such as the liver or the breast, which practitioners clinically palpate already, it is somewhat fortunately highly challenging to trick the natural protective barriers of remote organs such as the brain. So far, mechanical waves have been induced in the latter by shaking the surrounding cranial bones. Here, the skull was circumvented by guiding pressure waves inside the subject's buccal cavity so mechanical waves could propagate from within through the brainstem up to the brain. Repeatable, reproducible and robust displacement fields were recorded in phantoms and in vivo by magnetic resonance elastography with guided pressure waves such that quantitative mechanical outcomes were extracted in the human brain.     

Dynamic expression patterns of RhoV/Chp and RhoU/Wrch during chicken embryonic development.

Rho GTPases play central roles in the control of cell adhesion and migration, cell cycle progression, growth, and differentiation. However, although most of our knowledge of Rho GTPase function comes from the study of the three classic Rho GTPases RhoA, Rac1, and Cdc42, recent studies have begun to explore the expression, regulation, and function of some of the lesser-known members of the Rho GTPase family. In the present study, we cloned the avian orthologues of RhoV (or Chp for Cdc42 homologous protein) and RhoU (or Wrch-1 for Wnt-regulated Cdc42 homolog-1) and examined their expression patterns by in situ hybridization analysis both during early chick embryogenesis and later on, during gastrointestinal tract development. Our data show that both GTPases are detected in the primitive streak, the somites, the neural crest cells, and the gastrointestinal tract with distinct territories and/or temporal expression windows. Although both proteins are 90% identical, our results indicate that cRhoV and cRhoU are distinctly expressed during chicken embryonic development.     

Senescence impairs successful reprogramming to pluripotent stem cells

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by overexpressing combinations of factors such as Oct4, Sox2, Klf4, and c-Myc. Reprogramming is slow and stochastic, suggesting the existence of barriers limiting its efficiency. Here we identify senescence as one such barrier. Expression of the four reprogramming factors triggers senescence by up-regulating p53, p16^INK4a, and p21^CIP1. Induction of DNA damage response and chromatin remodeling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells.     

Osteocyte dendrogenesis in static and dynamic bone formation: an ultrastructural study

The present ultrastructural investigation into osteocyte dendrogenesis represents a continuation of a previous study (Ferretti et al., Anat. Embryol., 2002; 206:21-29), in which we pointed out that, during intramembranous ossification, the well-known dynamic bone formation (DBF), performed by migrating osteoblast laminae, is preceded by static bone formation (SBF), in which cords of stationary osteoblasts transform into osteocytes in the same site where they differentiated. The research was carried out on the perichondral center of ossification surrounding the mid shaft level of various long bones of chick embryos and newborn rabbits. Transmission electron microscope observations showed that the formation of osteocyte dendrites is quite different in the two types of osteogenesis, mainly depending on whether or not osteoblast movement occurs. In DBF, osteoblasts transform into small ovoidal/ellipsoidal osteocytes and their dendrites form in an asynchronous and asymmetrical manner in concomitance with, and depending on, the advancing mineralizing surface and the receding osteogenic laminae. In SBF, stationary osteoblasts give rise to big globous osteocytes, located inside confluent lacunae, with short and symmetrical dendrites that can radiate simultaneously all around their cell body because they are completely surrounded by unmineralized matrix. Contacts and gap junctions were observed between all osteocytes (both SBF- and DBF-derived) and between osteocytes and osteoblasts. Finally, a continuous osteocyte network extends throughout the bone, regardless of its static or dynamic origin. This network has the characteristic of a functional syncytium, potentially capable of modulating, by wiring transmission, the cells of the osteogenic lineage covering the bone surfaces.     

Energetics of running in top-level marathon runners from Kenya

On ten top-level Kenyan marathon runners (KA) plus nine European controls (EC, equivalent to KA), we measured maximal oxygen consumption ([Formula: see text]) and the energy cost of running (C (r)) on track during training camps at moderate altitude, to better understand the KA dominance in the marathon. At each incremental running speed, steady-state oxygen consumption ([Formula: see text]) was measured by telemetric metabolic cart, and lactate by electro-enzymatic method. The speed requiring [Formula: see text] provided the maximal aerobic velocity (v (max)). The energy cost of running was calculated by dividing net [Formula: see text] by the corresponding speed. The speed at lactate threshold (v (ΘAN)) was computed from individual La(b) versus speed curves. The sustainable [Formula: see text] fraction (F (d)) at v (ΘAN) (F (ΘAN)) was computed dividing v (ΘAN) by v (max). The F (d) for the marathon (F (mar)) was determined as F (mar)?=?0.92 F (ΘAN). Overall, [Formula: see text] (64.9?±?5.8 vs. 63.9?±?3.7?ml?kg(-1)?min(-1)), v (max) (5.55?±?0.30 vs. 5.41?±?0.29?m?s(-1)) and C (r) (3.64?±?0.28 vs. 3.63?±?0.31?J?kg(-1)?m(-1)) resulted the same in KA as in EC. In both groups, C (r) increased linearly with the square of speed. F (ΘAN) was 0.896?±?0.054 in KA and 0.909?±?0.068 in EC; F (mar) was 0.825?±?0.050 in KA and 0.836?±?0.062 in EC (NS). Accounting for altitude, running speed predictions from present data are close to actual running performances, if F (ΘAN) instead of F (mar) is taken as index of F (d). In conclusion, both KA and EC did not have a very high [Formula: see text], but had extremely high F (d), and low C (r), equal between them. The dominance of KA over EC cannot be explained on energetic grounds.     

Distribution and identity of neurons expressing the oxytocin receptor in the mouse spinal cord

Nurr1 is a member of the nuclear receptor superfamily and is a regulatory factor of differentiation, migration and maturation of mesencephalic dopaminergic neurons. The present study was designed to observe the dynamic changes in the protein expression of Nurr1 and the relationship between Nurr1 and proliferating cell nuclear antigen (PCNA) during rat brain and spinal cord development. And we also investigated the significance of Nurr1 in differentiation and migration of nerve cells. Paraffin-embedded sections, immunohistochemistry, immunohistochemical double staining and Western blot techniques were used. The results demonstrate that the presence of Nurr1-positive cells increased during embryo development and that these cells slowly migrated to locations far from the lateral ventricle. In postnatal rats, the presence of Nurr1-positive cells surrounding the lateral ventricle decreased markedly. The expression of Nurr1 in the cerebral cortex peaked at postnatal days 1-5 (P1-P5) and then decreased as the cells matured, becoming rare in the mature cerebral cortex. As the cells matured, a staircase-shaped migration of Nurr1-positive cells from dorsal areas to ventral areas of the spinal cord could be observed. As maturation continued, the presence of Nurr1-positive cells in the spinal cord decreased, and no Nurr1-positive cells were found in the mature spinal cord. The comparative observation of Nurr1 and PCNA showed that the two proteins were expressed in different regions and in different cells. Nurr1 was confined to differentiated and migrating immature cells and was not present in proliferating cells. We suggest that Nurr1 may play a regulatory role in the differentiation, migration and maturation of nerve cells in the rat brain and spinal cord.